The present invention relates to a monoclonal antibody specific to human L-PGDS present predominantly in cerebrospinal fluid (CSF), a hybridoma producing said monoclonal antibody, methods for detection of L-PGDS or diseases by said monoclonal antibody, and a kit for detection of L-PGDS by said monoclonal antibody.
Prostaglandin D is a biologically active substance synthesized in animal tissues from arachidonic acid released from a biomembrane upon stimulation, and it is produced from prostaglandin H (a common precursor of prostaglandin families produced by cyclo-oxygenase) by prostaglandin D synthase (PGDS).
The presence of two types of PGDS, glutathione-independent L-PGDS and glutathione-dependent spleen type PGDS, is known (Shimizu et, al., J. Biol. Chem., 254, 5222-5228 (1979); Urade et al., J. Biol. Chem., 260, 12410-12415 (1985); Christ-Hazelhof and Nugteren, Biochim. Biophys. Acta, 572, 43-51 (1979); Urade et al., J. Biol. Chem., 262, 3820-3825 (1987). The former is known to be predominantly located in the central nervous system such as brain, epididymis, spinal cord, retina, and inner ear (Urade et al., J. Biol. Chem., 260, 12410-12415 (1985); Ueno et al., J. Neurochem., 45, 483-489 (1985); Urade et al., J. Biol. Chem., 262, 3820-3825 (1987); Goh et al., Biochim. Biophys. Acta, 921, 302-311 (1987); and Tachibana et al., Proc. Natl. Acad. Sci. USA, 84, 7677-7680 (1987)), and the latter is known to be distributed broadly in almost all peripheral organs including spleen, bone marrow, digestive organs, thymus, and skin (Ujihara et al., Arch. Biochem. Biophys., 260, 521-531 (1988); and Ujihara et al., J. Invest., Dermatol., 90, 448-451 (1988)).
On the other hand, a protein called xcex2-trace was observed to be present specifically in human CSF, but its physiological function remained unrevealed (Causen, J. Proc. Soc. Exp. Biol. Med., 107, 170-172 (1961)).
A certain correlation between xcex2-trace known as a protein specific to CSF and severe brain disorders or certain diseases (multiple sclerosis, brain tumors, Meckel""s syndrome and paraproteinemia) was noted from the observation that xcex2-trace levels depend on such disorders (Ericsson et al., Neurology, 19, 606-610 (1969); Olsson et al., J. Neurol. Neurosurg. Psychiat. 37, 302-311 (1974); Link, J. Neurol. Sci., 16, 103-114 (1972); Whistsed and Penny, Clinica Chimica Acta, 50, 111-118 (1974); and Chemke et al., Clinical Genetics, 11, 285-289 (1977)). However, the exact correlation between xcex2-trace and such disorders could not be determined because the physiological function of xcex2-trace still remained unrevealed and because there was no tool available for determining the exact amount (concentration) of xcex2-trace.
Recently, the nucleotide sequence of cDNA coding for L-PGDS was reported (Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)), and production of L-PGDS by genetic recombination became feasible. The L-PGDS thus produced was examined and its amino acid sequence was estimated and compared with an N-terminal partial amino acid sequence of human xcex2-trace in searching for their homology (Kuruvilla et al., Brain Research, 565, 337-340 (1991), Zahn et al., Neuroscience Letters, 154, 93-95 (1993)) or with the amino acid sequence of purified human xcex2-trace (Hoffmann et al., J. Neurochem., 61(2), 451-456 (1993)), and further immunological examination was made using polyclonal antibodies (Watanabe et al., Biochem. Biophys. Res. Communication, 203, 1110-1116 (1994)). These studies revealed that xcex2-trace was identical with L-PGDS.
Prostaglandin D occurring abundantly in the central nervous system functions, in one physiological action, as a neuromodulator of several central actions including sleep promotion. Prostaglandin D synthase is considered as a key enzyme for sleep-wake activities (Hayashi, FASEB J., 5, 2575-2581 (1991)), and it is believed that at least a part of the L-PGDS secreted from competent cells is accumulated in CSF (Watanabe et al., Biochem. Biophys. Res. Communication, 203, 1110-1116 (1994)).
Accordingly, the analysis of L-PGDS distribution etc. in the central nervous system is useful for detection of diseases in the central nervous system, and it is expected that L-PGDS levels in CSF or humor can also be used as an indicator in early diagnosis and prognostic observations for other diseases caused by abnormalities in the central nervous system. It is further expected that L-PGDS (or xcex2-trace) can be used for examination of a reproduction ability, diagnosis of fetal growth, etc. because this enzyme is distributed in such humors derived from genital organs, as semen, oviduct fluid and amniotic fluid, as well. For such applications, there is demand for antibodies specifically recognizing L-PGDS.
Nevertheless, such antibodies have still not been established with high specificity to meet such demand.
The object of the present invention is to provide a monoclonal antibody specifically recognizing L-PGDS, a hybridoma producing said antibody, methods for detection of L-PGDS or diseases by said monoclonal antibody, and a kit for detection of L-PGDS by said monoclonal antibody.
As a result of their eager researches, the present inventors have successfully obtained a monoclonal antibody specifically recognizing L-PGDS from a hybridoma prepared by cell fusion between myeloma cells and antibody-producing cells from an animal immunized with L-PGDS which is a major protein in human CSF, and they thereby arrived at the present invention.
That is, the present invention relates to a monoclonal antibody specifically recognizing L-PGDS. The subclass of such monoclonal antibody includes immunoglobulin G1 or G2.
Further, the present invention relates to a hybridoma producing said monoclonal antibody by cell fusion between myeloma cells and antibody-producing cells from an animal immunized with L-PGDS.
Further, the present invention relates to methods for detection of L-PGDS or diseases by said monoclonal antibody. An example of such diseases is oligospermia.
Further, the present invention relates to a kit for detection of L-PGDS, which is selected from a reagent containing said monoclonal antibody labeled with an enzyme and a substrate solution, and a reagent containing said monoclonal antibody obtained by biotination, enzyme-labeled avidin, and a substrate.
Further, the present invention relates to a method for detection of diseases by said kit for detection of L-PGDS. An example of such diseases is oligospermia.
Hereinafter, the present invention is described in detail.
Production of the present monoclonal antibody against L-PGDS consists of the steps of:
(1) Preparation of an antigen;
(2) Immunization and preparation of antibody-producing cells;
(3) Establishment of an antibody-titration system;
(4) Cell fusion;
(5) Selecting and cloning hybridomas; and
(6) Isolation of the monoclonal antibody.
Hereinafter, each step is described.
(1) Preparation of an Antigen
L-PGDS can be produced in large amounts in a usual manner by E. coli and CHO cells etc. with its known cDNA (Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)). In this production of L-PGDS, a recombinant DNA containing the cDNA for L-PGDS is constructed and transformed into a microorganism, which is then cultured to produce the enzyme. The L-PGDS thus produced can be purified from the culture by conventional means.
Then, an immunogen is prepared by dissolving the resulting L-PGDS in a buffer and then adding an adjuvant to it. Examples of such adjuvants are Freund complete adjuvant, Freund incomplete adjuvant, BCG, Hunter""s Titermax (CytRx Corporation), key hole limpet hemocyanin-containing oil, etc., and any of them can be mixed.
(2) Immunization and Preparation of Antibody-producing Cells
The immunogen thus obtained is administrated as antigen into mammals such as horse, monkey, dog, pig, cow, goat, sheep, rabbit, guinea pig, hamster and mouse, or birds such as pigeon and chicken. In particular, mouse, rat, guinea pig, rabbit and goat are preferably used. Any of the known immunization methods may be employed preferably using i.v., s.c., or i.p. administration. Immunization intervals are not particularly limited, and the immunogen is given 2 to 10 times, preferably 2 to 5 times, preferably at intervals of several days to several weeks, more preferably 1 to 3 weeks.
1 to 10 days preferably 2 to 5 days after the final immunization, antibody-producing cells are prepared from the animal. Examples of such antibody-producing cells are spleen cells, lymph node cells, thymocytes and peripheral blood cells, and generally spleen cells are used conventionally. In the case of mouse, 0.01 xcexcg to 1,000 xcexcg, preferably 1 to 300 xcexcg antigen is given per animal in one administration.
(3) Establishment of an Antibody-titration System
It is necessary to establish a system of measuring the antibody titer in serum from the immunized animal or in a culture supernatant from the antibody-producing cells, so that the immune response level of the immunized animal can be confirmed and the desired hybridoma can be selected from the fusion cells. For example, the antibody can be detected conventionally using known methods such as enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay (FIA). Although the present method for detecting the antibody is not limited to the above methods, ELISA is conveniently used for easy operations. Usually, L-PGDS is put to each well of a 96-well plastic microtiter plate and left at room temperature to immobilize the enzyme onto the well. Then, the unbound sites of the L-PGDS as antigen are blocked with calf serum albumin, fetal bovine serum, skim milk, or gelatin. Then, an antiserum diluted with phosphate buffered saline (referred to hereinafter as PBS) or a culture supernatant of the hybridoma is added to each well. Subsequently, commercial secondary antibody labeled with an enzyme or fluorescent compound or with biotin is added to each well, followed by adding a coloration substrate. The coloration occurring can be determined in a photometer or fluorometer to quantify the antibody against L-PGDS.
(4) Cell Fusion
The myeloma cells to be subjected to cell fusion with the antibody-producing cells are those derived from animal species such as mouse, rat, and human, easily available to those skilled in the art. The cell line used is preferably drug-resistant, which upon fusion with the antibody-producing cells, is rendered survivable in a selection medium such as HAT medium. A cell line resistant to 8-azaguanine is generally used. Because such cell line lacks hypoxanthine-guanine phosphoribosyltransferase (HGPRT-), it can not grow in hypoxanthine-aminopterine-thymidine (HAT) medium. The cell line used is preferably cells not secreting immunoglobulins.
Examples of such myeloma cells are mouse myeloma cell lines such as P3X63Ag8 (ATCC TIB-9; Nature, 256, 495-497 (1978)), P3X63Ag8U.1 (P3U1) (ATCC CRL-1580; Current Topics in Microbiology and Immunology, 81, 1-7 (1978), P3X63Ag8.653 (ATCC TIB-18; European J. Immunology, 6, 511-519 (1976)), and P2/NSI/1-Ag4-1 (ATCC CRL-1581; Nature, 276, 269-270 (1978)); rat myeloma cell lines such as for example 210.RCY.Agl.2.3 (Y3-Agl.2.3) (ATCC CRL-1631; Nature, 277, 131-133 (1979)); human myeloma cell lines such as U-266-AR1 (Proc. Natl. Acad. Sci. USA, 77, 5429 (1980)), GM 1500 (Nature, 288, 488 (1980)), and KR-4 (Proc. Natl. Acad. Sci. USA, 79, 6651 (1982)).
The antibody-producing cells are obtained from spleen cells, lymph node cells, thymocytes, and peripheral blood cells. Briefly, the antibody-producing cells are prepared as follows: Tissues such as spleen, lymph node or thymus are excised or blood are collected from the immunized animals. These tissues are disrupted and then suspended in a buffer such as PBS or in a medium such as DMEM, RPMI 1640 and E-RDF. This cell suspension is filtered through e.g. a #200-250 stainless mesh and then centrifuged to give the desired antibody-producing cells.
Then, the antibody-producing cells are subjected to cell fusion with myeloma cells.
Before cell fusion, myeloma cells suitable for antibody production are selected. 106 to 108 cells/ml antibody-producing cells are mixed with 106 to 108 cells/ml myeloma cells at a ratio of from 1:1 to 1:10 in an animal cell growth medium, e.g. Eagle""s minimal essential medium (MEM), Dulbecco""s modified Eagle""s medium (DMEM), RPMI-1640 medium, or E-RDF medium. To enhance cell fusion, these cells are mixed at a ratio of e.g. 1:6 and then incubated for 1 to 15 minutes in the presence of a fusion enhancer in e.g. RPMI-1640 medium containing dimethylsulfoxide at a temperature of 30 to 37xc2x0 C. This fusion enhancer may be polyethylene glycol with an average molecular weight of 1,000 to 6,000, polyvinyl alcohol or Sendai virus. Alternatively, the antibody-producing cells can be subjected to cell fusion with myeloma cells by electric stimulation (e.g. electroporation) in a commercial cell fusion apparatus.
(5) Selection and Cloning of Hybridomas
After cell fusion, the cells are screened for desired hybridomas. The selective growth of the fusion cells in a selection medium may be used for screening as follows: The cell suspension is diluted 5- to 10-fold with e.g. E-RDF medium containing 15% fetal bovine serum and then put to each well of a microtiter plate at about 102 to 106 cells/well, followed by addition of a selection medium (e.g. HAT medium) to each well. Thereafter, the cells are incubated while the selection medium is exchanged with fresh one at suitable intervals.
Where myeloma cells are from an 8-azaguanine-resistant strain and HAT medium is used as selection medium, the myeloma cells and antibody-producing cells, if they fail to fuse, will die during in vitro culture in about 7 days and antibody-producing cells in 10 days. Hence, hybridomas can be obtained from those cells beginning to grow after the 10th day of culture.
The hybridomas are screened for the desired ones by examining their supernatants on the presence of the antibodies against L-PGDS. This screening step can be carried out by any of the conventional methods. For example, a supernatant (first antibody) from hybridomas grown in each well is put to a well with L-PGDS immobilized on it, then a labeled secondary antibody is added to the well and incubated, and the binding ability of the secondary antibody is then examined in enzyme immunoassays (EIA, ELISA), RIA, etc.
In more detail, the screening of hybridomas is carried out as follows: Natural or recombinant L-PGDS, which was used as immunogen, is immobilized as antigen onto a 96-well microtiter plate. A culture supernatant expected to contain the monoclonal antibody is added to each well and reacted with the immobilized antigen. Then, the antigen-bound monoclonal antibody, if any, is reacted with another antibody (enzyme-labeled anti-immunoglobulin antibody). Alternatively, said immobilized monoclonal antibody is reacted with a biotinylated anti-immunoglobulin antibody and then with enzyme-labeled avidin. Finally, each well is colored by adding an enzyme substrate solution. The hybridomas whose culture supernatants are colored in the wells having the immobilized natural or recombinant L-PGDS are those producing antibodies having the ability to bind to the L-PGDS.
These hybridomas can be cloned in conventional methods including limiting dilution, soft agar cloning, fibrin gel cloning and fluorescence excitation cell sorting to give the desired monoclonal antibody-producing hybridoma.
(6) Isolation of the Monoclonal Antibody
From the resulting hybridoma, the monoclonal antibody can be isolated using conventional methods such as cell culture method, ascites transudate method, etc.
In the cell culture method, the hybridoma is cultured for 2 to 14 days in a medium such as RPMI-1640, MEM, or E-RDF containing 10 to 20% calf serum or in a serum-free medium under conventional culture conditions, e.g. 37xc2x0 C., 5% CO2. The antibody can be obtained from the culture.
In the ascites transudate method, a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is administrated by i.p. to the same mammal species as the mammal from which the myeloma cells were derived. Then, the hybridoma, 1xc3x97107 to 1xc3x97109 cells, preferably 5xc3x97107 to 1xc3x97108 cells, are administrated by i.p. to the animal, and a large amount of hybridoma cells are grown in the animal. After 1 to 4 weeks, preferably 2 to 3 weeks, ascites fluid or serum is collected from the animal.
If it is necessary to purify the antibody from the ascites fluid or serum, it can be purified by conventional methods such as salting-out with ammonium sulfate, ion-exchange chromatography on anion exchanger e.g. DEAE cellulose, affinity chromatography on Protein A SEPHAROSE, and gel filtration, and these may be used singly or in combination.
The method of detecting L-PGDS according to the present invention can be carried out using said monoclonal antibody, as follows:
A 96-well microtiter plate is coated with a diluted sample such as CSF, serum etc. and then blocked with e.g. 0.2% gelatin in PBS. Then, the monoclonal antibody of the present invention, labeled with an enzyme, is added to each well and then incubated; alternatively, the monoclonal antibody labeled with biotin is added to each well, then the plate is washed, enzyme-labeled avidin or streptoavidin is added to each well, and the plate is further incubated. Then, the plate is washed and a coloration substrate such as ABTS (2,2xe2x80x2-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)) is added to each well. L-PGDS can be determined by examining this coloration in the calorimetric method.
In another embodiment of the present invention, a 96-well microtiter plate is coated with the diluted monoclonal antibody of the present invention and then blocked with e.g. 0.2% gelatin in PBS. Then, a diluted sample such as CSF, serum etc. is added to each well and the plate is incubated. After washing the plate, another enzyme-labeled monoclonal or polyclonal antibody solution is added to each well and the plate is incubated; alternatively, the monoclonal antibody or polyclonal antibody labeled with biotin is added to each well, then the plate is washed, enzyme-labeled avidin or streptoavidin is added to each well, and the plate is further incubated. Then, the plate is washed and a coloration substrate such as ABTS (2,2xe2x80x2-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)) is added to each well. The L-PGDS can be determined by examining this coloration in the calorimetric method. In this manner, it is possible to detect and quantify L-PGDS.
The monoclonal antibody of the present invention is useful as a reagent for measurement of L-PGDS because it specifically binds to L-PGDS. Furthermore, the present monoclonal antibody is useful as a reagent in determining the presence and distribution of not only L-PGDS as antigen but also other similar antigens having the same epitope as that of L-PGDS, as well as fragments of L-PGDS in biological samples such as organs, tissues, cells and humor. Hence, the antibody of the present invention is useful as a reagent for such measurement and diagnosis. The detection or measurement of L-PGDS in organs, tissues, cells and humor can be effected using quantitative or qualitative means such as EIA, ELISA, RIA, FIA, Western blot technique and immunohistochemistry, etc.
The kit of the present invention, if an enzyme is used as a label for detection, contains the following ingredients:
(1) monoclonal antibody labeled with an enzyme; and
(2) substrate.
The kit of the present invention, if modified with the sandwich ELISA method, contains the following ingredients:
(1) monoclonal antibody;
(2) monoclonal or polyclonal antibody labeled with an enzyme; and
(3) substrate.
The kit of the present invention, if modified with the biotin-avidin method, contains the following ingredients:
(1) biotinated monoclonal antibody;
(2) enzyme-labeled avidin or streptavidin; and
(3) substrate.
The kit of the present invention, if modified with the sandwich ELISA and biotin-avidin methods, contains the following ingredients:
(1) monoclonal antibody;
(2) biotinated monoclonal or polyclonal antibody;
(3) enzyme-labeled avidin or streptavidin; and
(4) substrate.
In the ingredients, the xe2x80x9cmonoclonal antibodyxe2x80x9d means the monoclonal antibody of the present invention. The xe2x80x9cpolyclonal antibodyxe2x80x9d means an antibody contained in serum from an animal immunized with L-PGDS, and it can be prepared in the following manner.
(1) Preparation of the Antigen
L-PGDS can be produced in large amounts in a usual manner by E. coli and CHO cells etc. with its known cDNA (Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)). For production of L-PGDS, a recombinant DNA containing the cDNA for L-PGDS is constructed and transformed into a microorganism, and the transformant is cultured to produce the enzyme. The resulting L-PGDS can be purified from the culture by conventional means.
L-PGDS thus obtained is dissolved in a buffer, and an immunogen is prepared by adding an adjuvant to it. Examples of adjuvants are Freund complete adjuvant, Freund incomplete adjuvant, BCG, Hunter""s Titermax (CytRx Corporation), key hole limpet hemocyanin-containing oil, etc., and any of them can be mixed.
(2) Immunization and Preparation of Blood
The immunogen thus obtained is administrated into mammals such as horse, monkey, dog, pig, cow, goat, sheep, rabbit, guinea pig, hamster and mouse, or birds such as pigeon and chicken, among which mouse, rat, guinea pig, rabbit and goat are preferably used. Any of the known immunization methods can be employed preferably via i.v., s.c., or i.p. administration. Immunization intervals are not particularly limited, and this administration is carried out 2 to 10 times, preferably 2 to 5 times, at intervals of preferably several days to several weeks, more preferably 1 to 3 weeks.
Antibody titer in blood from the immunized animal is determined according to the above method in (3) xe2x80x9c1. Production of the monoclonal antibodyxe2x80x9d. Blood samples found to have high antibody titer are left at room temperature or 4xc2x0 C. and centrifuged to give serum containing the polyclonal antibody.
If it is necessary to purify the polyclonal antibody from the serum, it can be purified by conventional methods such as salting-out with ammonium sulfate, ion-exchange chromatography on anion exchanger e.g. DEAE cellulose, affinity chromatography on Protein A Sepharose, and gel filtration separating molecules depending on molecular weight and structure, and these may be used in singly or in combination.
According to the present invention, various diseases can be detected by use of the present kit for detection of L-PGDS. For example, oligospermia can be diagnosed readily and rapidly by the present kit using the monoclonal antibody.
The monoclonal antibody of the present invention can also be used to purify L-PGDS. That is, the monoclonal antibody of the present invention is coupled in a usual manner to carriers such as agarose, cellulose, acrylamide gel, commercially available self-made affinity carriers and then washed. L-PGDS can be purified easily with high yield by elution from the column with a suitable solvent or buffer.